Cytoexpert

Preparing samples in 96-well plates for flow cytometry. For transfection experiments, cells were transfected 2d before being harvested. Then, media was aspired, 100 μl PBS was added to wash the cells, the PBS was aspired, and 40 μl Trypsin-EDTA (GIBCO) was added. Following this, cells were suspended and transferred to 96-well plates. The plated were spun down at 200 × g, 5 min, and the media was aspirated. Cells were resuspended in 200 μl 4% paraformaldehyde (PFA, Boster Biological Technology). One hundred thousand cells were analyzed for each sample on a Beckman Coulter CytoFlex S flow cytometry or BD Fortessa SORP equipped with proper lasers and filters, and data were analyzed with CytoExpert (Beckman Coulter) or FlowJo (V10) software, respectively. Some of the flow cytometry data of Figs. 1–4 are given in Supplementary Materials (Supplementary Figs. 2, 4, and 9). Real-time qPCR was performed by RuiBiotech Co., Ltd. The primers used to quantify Citrine mRNA levels and CHO GAPDH (Cricetulus griseus) primers used for normalization were as follows:
GAPDH-CHO-F: 5’-GAAGGTGGTGAAGCAGGCAT;
GAPDH-CHO-R: 5’-CAGCCCCAGCATCAAAGGTG;
Citrine-F: 5’-TCAAGGACGACGGCAACTAC;
Citrine-R: 5’-GTCCTCCTTGAAGTCGATGC.

Synechocystis cells that were starved from nitrogen for ~2 weeks before FACS experiments. Cells were sorted with a MoFlo XDP (Beckman Coulter, Munich, Germany) into 500 µL PBS buffer using a 70 uM CytoNozzle at 30 p.s.i. and PBS [pH 7.0] as sheath. Before FACS or analysis, 1 µL of BODIPY (5 mg/mL) was added to 500 µL of cells and incubated for 10 min. Cells were identified based on their scatter (SSC-LA vs. FSC-LA), chlorophyll-fluorescence (670/30) captured from a 488 nm (70 mW) laser. Cells were divided into low and high producers based on their emission profile and at 534/30 (BODIPY) when compared to unstained and PHB deficient cells. For analysis, the software Summit FACS and FCS Express was used. BODIPY staining was also scored using a Cytoflex analyzer (Beckmann Coulter, Munich, Germany) equipped with a single 488 nm laser. Principle BODIPY emission was captured with a 525/40 bandpass and plotted against scatter to remove clumplets and distinguish BODIPY. PHB content was inferred when compared to unstained and PHB deficient cells. For analysis and illustration of the date, the software programs CytoExpert (Beckman Coulter, Munich, Germany) and FlowJo (FlowJo LLC, Oregon, USA) were used.

To determine the effect of VEGFa‐CXCR4 codelivery on differentiation of EPCs in vitro, 5 × 10⁵ EPCs transfected with SPION/PEG‐PEI/plasmid (C‐V, CXCR4, VEGFa and Vector) were induced to differentiate after 14 d of induction. The differentiation of EPCs was characterized by immunostaining for E‐selectin and VE‐cadherin expression, as described above. The endothelial differentiation of cells was observed using a fluorescence microscope (BX63; Olympus, Tokyo, Japan). The quantification of endothelial differentiation capacity was analyzed using Image J software. The percent of differentiation was calculated as the total number of nuclei present divided by the number of VE‐cadherin or E‐selectin positive cells. Untreated cells were used as control. The quantification of endothelial differentiated from EPCs was also validated using flow cytometry. Briefly, cells were collected by centrifugation, resuspended in 100 µL of FACS buffer containing 1 µg of the indicated antibody, dispensed in a minimum of 10⁵ cells per sample, gently mixed, and incubated on ice for 30 min. Flow cytometry (CytoExpert, Beckmann Coulter) and data analysis software Flow Jo software version 10.5 (TreeStar, Inc, Ashland, OR) were used.

Cell proliferation and cell cycle assays were performed as described above. In brief, for cell proliferation, 10³ cells were seeded in a 96‐well plate on day 0 with the treatment. Absorbances at 490 nm were measured using CellTiter 96 AQueous One Solution Cell Proliferation Assay kit (Promega) on day 2, day 4, and day 6 to measure the cellular proliferation. For cell cycle, cells were washed with PBS twice and resuspended in 1% formaldehyde buffer. After incubation with propidium for 30 min, samples were assessed with CytoExpert, Beckmann Coulter.

The 8-OHdG immunodetection assay was carried out on a CytoFlex S (Beckman Coulter, Inc.) equipped with violet (405 nm) and blue (488 nm) lasers for the excitation of Hoechst 33342 and Alexa Fluor 488, respectively. Alexa Fluor 488 has a maximum emission at 520 nm and a FITC photodetector (525/40 band-pass filter) was used, while Hoechst 33342 has a maximum emission at 461 nm and a PB450 photodetector (450/45 band-pass filter) was used. The flow cytometry data were analysed using the software CytoExpert version 2.3.0.84 (Beckman Coulter, Inc.). SCSA^® was carried out on a Cytomics FC-500 (Beckman Coulter, Inc.) equipped with a 488 nm argon ion laser for the excitation of AO. AO green fluorescence was detected with a 530/28 band-pass filter (FL-1), while AO red fluorescence was detected with a 620/40 band-pass filter (FL-3). The analysis of the data was carried out using the software WEASEL version 2.4. Non-sperm events, such as bacteria or extender particles, were discarded by gating in an FSC (forward scatter of the laser light)/SSC (side scatter of the laser light) dot plot based on differences in complexity and size among debris and sperm cells [28 (link)].